| PDZ domains are protein scaffolds that modulate a variety of cellular processes, holding their targets in the correct intracellular location for efficient signaling. G protein-activated phospholipase C-beta (PLC-beta) isozymes contain PDZ domain-binding sequences at their C-termini. Upon stimulation by receptor-coupled G-proteins, PLC-betas hydrolyze phosphotidylinositol bisphosphate to the ubiquitous second messengers inositol trisphosphate and diacylglycerol, which activate a host of downstream signaling cascades.;The Drosophila phototransduction cascade is a model system for the study of PLC-beta-mediated signaling. The retinal protein i&barbelow;nactivation n&barbelow;o a&barbelow;fter-potential D&barbelow; (InaD), containing five PDZ domains, is absolutely required for the proper targeting and retention of the PLC-beta isozyme no r&barbelow;eceptor p&barbelow;otential A&barbelow; (NorpA). When NorpA is mislocalized, the photoresponse is severely aberrant, highlighting the critical role of InaD in regulation of phototransduction. We determined the crystal structure of the N-terminal PDZ domain (PDZ1) of InaD in complex with the PDZ-binding sequence of NorpA, allowing a detailed structural analysis of how InaD interacts with NorpA. Specifically, the crystal structure revealed an intermolecular disulfide bond between PDZ1 and NorpA, suggesting that this high-affinity interaction is regulated by the redox potential of the retinal cell. In vitro and in vivo experiments showed that the disulfide bond is necessary for interaction between PDZ1 and NorpA, and that it formed even in the normally reducing intracellular environment.;Other PDZ-domain containing proteins have been implicated in binding PLC-beta isozymes in a more typical fashion that the disulfide-linked complex described above for InaD and NorpA. The N&barbelow;a+/H&barbelow; + e&barbelow;xchanger r&barbelow;egulatory f&barbelow;actor (NHERF) family of proteins, each containing two PDZ domains and a cytoskeletal binding domain, have been implicated in regulation of mammalian PLC-beta isozymes. In particular, the second PDZ domain of NHERF2 (PDZ2) has been shown to interact specifically with PLC-beta3, even though related PLC-beta isozymes contain similar PDZ domain binding sequences. Through in vitro analysis, we showed that NHERF2 PDZ2 interacts specifically with PLC-beta3 and not PLC-beta2. In addition, mutational analysis of both PDZ2 and PLC-beta3 revealed structural explanations for the preference of PDZ2 for PLC-beta3. These results may be extrapolated to explain the specificity type I PDZ domains in general. |
